Central to processes important in the pathogenesis of local stasis and thrombosis in the pulmonary vasculature in sickle cell disease (SCD) is the vascular endothelium and alterations in its metabolic functions. Using ex vivo model of SCD, we have observed that exposure to plasma from patients with acute chest syndrome (ACS) causes a ten-fold increase in nitrogen oxides (NO2) from cultured pulmonary artery endothelial cell (PAEC). In subsequent studies, we found: 1) this increase in NO, is associated with increased eNOS protein expression and enzymatic activity but not with a change in transcripts of constitutive nitric oxide synthase (NOSIII) nor any change in iNOS components; 2) a marked reduction in PAEC antioxidant molecules; 3) increased production of PAEC superoxide; 4) substantial formation of ONOO- both ex vivo and in lungs of a transgenic sickle cell mouse during induced ACS; and 5) an increase in markers of oxidative stress (F2:-isoprostanes) in plasma of patients during ACS. These findings suggest that NO, (including ONOO-) production and the oxidative state into which it is secreted contribute to the cellular and tissue injury observed in acute, as well as chronic, pulmonary disease associated with SCD. To investigate this hypothesis in vivo, we will focus on: 1) defining the role of NO in vascular responses and vasoocclusive events in a sickle cell transgenic mouse by correlating functional assays, lung histology and animal mortality with changes in NO metabolism at baseline and during crisis induced by hypoxia: 2) defining the effect of pharmacological manipulation of ACS-like crisis in a sickle cell transgenic mouse using: a) NOS substrate, NOS cofactors, NOS inhibitors and inhaled NO or antioxidants and examining functional assays, lung histology and animal mortality at baseline and during crisis induced by hypoxia: and 3) defining the importance of NO metabolism and oxidative stress in a sickle cell transgenic mouse by interbreeding it with: a) a NOSIII (or NOll) deficient mouse; b) a glutathione peroxidase (GPI) deficient mouse: or c) a glucose-6-phosphate dehydrogenase (G6PD) mutant mouse and examining functional assays, lung histology and animal mortality at baseline and during induced crisis. These studies will investigate the contribution of altered NO metabolism and the oxidative state to the pathogenesis of the acute and chronic pulmonars." disease associated with SCD. These studies may also prove germane to extra-pulmonary VOC and may lead to therapies to prevent or reverse EC activation/dysfunction that appears central to this disease process.